细粒棘球蚴重组14-3-3基因的表达、纯化及免疫学鉴定

目的 构建细粒棘球蚴14-3—3基因的重组质粒并原核表达、纯化该重组蛋白，对其免疫学特性进行初步鉴定。方法 从重组质粒pGEM—T／Eg14-3—3中获取14—3—3基因，亚克隆于表达载体pET28a构建基因丁程菌株，并表达、纯化重组蛋白，经Westernblot、ELISA对该蛋白的免疫学特性进行初步研究。结果 成功构建含目的片段14—33的基因工程菌株；ELISA检测显示，用表达、纯化的重组蛋白免疫小鼠，诱导产生了特异性抗体，Westernblot鉴定该抗体能识别重组抗原及原头蚴、囊液、囊壁抗原。结论构建的pET28a／Eg14—3—3菌株能高效表达14—3—3蛋白，初步鉴定该重组蛋白具有较好的抗原性和免疫原性。

Expression, purification and immunologic identification of the recombinant 14-3-3 gene from Echiococcus granulosus(Chinese mainland strain)

Objective To construct Eg14-3-3 gene and express prokaryotically it, to purify and identify the immunogenicity of recombinant 14-3-3 protein. Methods Egl4 3 3 gene was attained from pGEM T/Eg14-3-3 recombinant plasmid and subcloned into high-level expressed vector pET28a. Eg14-3-3/pET28a/BL21plys was constructed. Western blot and ELISA were used to identify the immunogenicity of the recombinant protein. Results Egl4 3 3/pET28a/BL21plys recombinant plasmid was constructed. ELISA indicated that 14-3 3-immunized mice could produce the specific antibody; Western blot analysis showed that recombinant protein and protoscolex, cystic fluid and cystic wall of Echiococcus granulosus. could be recognized by this antibody. Conclusion pET28a/Eg14-3-3 recombinant plasmid is constructed and expressed efficiently. The antigenicity and immunogenicity of Eg14-3-3 protein is identified.